Pig Intestinal Mucosa Research Articles

Absorption of Vitamin B12 by the Guinea‐Pig I. SUBCELLULAR LOCALIZATION OF VITAMIN B12 IN THE ILEAL ENTEROCYTE DURING ABSORPTION*

Summary.Guinea‐pig intestinal mucosa was fractionated into brush borders, nuclei, mitochondria, lysosomes, microsomes and a soluble fraction; the mitochondria were subfractionated into inner and outer membranes. Vitamin B12, assayed micro‐biologically using Euglena gracilis, was found to be predominantly localized to the inner mitochondrial membranes. In the fasted animal, the concentration of vitamin B12 was similar in the jejunum and ileum.Approximately 25% of 10 ng of [58Co]cyanocobalamin given orally was retained after 7 days, when measured by a whole‐body counting technique.Fasted guinea‐pigs were fed 8–11 ng of [57Co]cyanocobalamin and the distribution of radioactivity throughout the gastro‐intestinal tract was studied. Although radioactivity was present in the ileal mucosa within 1 hr of feeding, no significant hepatic activity was present until 3 hr, confirming the presence of a mucosal delay in vitamin B12 absorption, a characteristic finding of intrinsic factor mediated vitamin B12 absorption. Seventy per cent of the vitamin B12 was found in the colon at 3 and 4 hr indicating that approximately 30% of the dose was absorbed.The ileal mucosa showed a highly significant rise in mitochondrial specific activity of labelled vitamin B12 during the first 2 hr post‐feeding. This subsequently decreased coincidentally with the rise in hepatic activity. It is suggested that the mitochondria play a hitherto unsuspected role in vitamin B12 absorption. No significant lysosomal radioactivity occurred during the absorptive period; this observation does not support the suggestion that vitamin B12 is absorbed by pinocytosis.

Phosphoenolpyruvate-activated phosphorylation of sugars by intestinal mucosa

1. 1. Guinea pig and rat intestinal mucosa were evaluated for the presence of a phosphoenolpyruvate (PEP): monosaccharide phosphotransferase system. A PEP-activated phosphotransferase system was found in the soluble fraction of intestinal' mucosa which differed from ATP-dependent phosphotransferase activity. Its kinetic parameters were evaluated. 2. 2. In contrast to the ATP-dependent system and to an ATP-generating enzyme, pyruvate kinase, the PEP-activated system was labile even when stored at −18° and was very sensitive to heating at 45° and to trypsin. However, the PEP-activated system was not affected by KF, whereas intestinal pyruvate kinase was completely inhibited at 15 mM KF. 3. 3. The PEP-activated system phosphorylated fructose only to fructose 1-phosphate, while the ATP-dependent system yielded the three expected products, fructose 1-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. In addition, the activity with ATP and PEP together in one assay equalled the sum of the separate ATP-dependent and PEP-activated systems. In experiments with [ 32P]PEP, concentrations of unlabeled ATP above 10 mM produced no dilution of enzymatically labeled N-acetylglucosamine 6-[ 32P]phosphate. 4. 4. The PEP-activated system in the intestinal mucosa had no apparent membrane component and did not phosphorylate 3- O-methylglucose. To date, no evidence has been obtained to indicate that this newly described PEP-activated system is related to intestinal sugar transport.

Conversion of Dihydrosphingosin to Palmitaldehyde and Palmitic Acid with Cell-free Preparations of Guinea Pig Intestinal Mucosa.

Conversion of Dihydrosphingosin to Palmitaldehyde and Palmitic Acid with Cell-free Preparations of Guinea Pig Intestinal Mucosa.

The subcellular localization of pteroyl polyglutamate hydrolase and folate in guinea pig intestinal mucosa.

1.1. Guinea pig intestinal mucosa was fractionated by two methods and the subcellular localization of pteroyl polyglutamate hydrolase (formerly known as folate conjugase) was determined.2.2. Pteroyl polyglutamate hydrolase appeared to be localized predominantly in the lysosomal fraction: no significant brush-border localization of the enzyme was noted.3.3. Folate was concentrated in the mitochondrial fraction.4.4. Possible mechanisms of pteroyl polyglutamate absorption are discussed in the light of the findings. 1. Guinea pig intestinal mucosa was fractionated by two methods and the subcellular localization of pteroyl polyglutamate hydrolase (formerly known as folate conjugase) was determined. 2. Pteroyl polyglutamate hydrolase appeared to be localized predominantly in the lysosomal fraction: no significant brush-border localization of the enzyme was noted. 3. Folate was concentrated in the mitochondrial fraction. 4. Possible mechanisms of pteroyl polyglutamate absorption are discussed in the light of the findings.

Folic acid conjugase: inhibition by unconjugated dihydroxy bile acids.

SummaryEvidence is presented to show inhibition of guinea pig intestinal mucosa gamma glutamyl carboxypeptidase (conjugase) by dihydroxy bile acids. Cholic acid, a trihydroxy acid, shows minimal inhibition at physiologic levels, while chenodeoxycholic and deoxycholic acids show striking inhibition.

Intestinal vitamin B12 absorption in the dog

Summary The aim of this study was to determine whether the intestinal absorption of vitamin B 12 in the dog is mediated by intrinsic factor. Dog gastric juice and duodenal mucosal extracts were tested for their ability to promote intestinal absorption of radioactive vitamin B 12 in vitro on guinea pig and dog intestinal mucosa homogenates and everted dog jejunal and ileal sacs. The absorption by in situ dog ileal sacs of radio B 12 alone and radio-B 12 bound to dog gastric juice or duodenal mucosal extracts into blood and liver was further studied in vivo by means of a double label isotope technique. In all systems the absorption of B 12 alone was higher than that of B 12 bound to dog gastric juice or duodenal mucosal extracts, which rather inhibited B 12 absorption. These studies do not support the notion that dog gastric juice and duodenal mucosal extracts are endowed with intrinsic factor activity and that, in the dog, physiological B 12 absorption in the intestine is mediated by intrinsic factor.

Intestinal vitamin B12 absorption in the dog

Summary The secretory, immunological, electrophoretic, chromatographic, and physiological properties of the vitamin B 12 binders in dog gastric juice and dog gastrointestinal mucosal extract were studied to determine whether they resembled intrinsic factor of other species. Total B 12 binding capacity of histamine- and urecholine-stimulated dog fundic gastric juice was much higher than that reported for man. Extracts of fundic gastric mucosa showed B 12 binding capacity far higher than that of dog antrum or intestine, indicating that the fundus is the main site of the B 12 binders secretion in the dog. Paper electrophoresis of dog fundic gastric juice revealed a single B 12 binder whose mobility and isoelectric point differed from that of human intrinsic factor, and which was highly susceptible to peptic digestion. When the B 12 binding materials of fundic gastric juice were separated by sequential chromatography on Sephadex G-25, Amberlite IRC-50, and Sephadex G-100 columns, two B 12 binders were obtained, whose electrophoretic mobilities differed from those of human intrinsic factor. These two binders were also inactive on guinea pig and dog intestinal mucosa homogenate assay for intrinsic factor. The secretion of B 12 binders in dog gastric juice, following histamine and urecholine stimulation, also showed a different pattern from that of intrinsic factor in man. Finally, none of the B 12 binders from dog gastric juice or mucosal extracts cross-reacted with the antibody to human intrinsic factor contained in pernicious anemia serum. These results indicate that the B 12 binders of dog gastric juice and dog gastrointestinal mucosa have none of the secretory, immunological, physicochemical, and physiological properties of intrinsic factor in other species but are rather similar to the "tertiary" B 12 binders related to mucosubstances of the gastrointestinal tract.

The presence of sphingomyelin- and ceramide-cleaving enzymes in the small intestinal tract

The crude protein fraction of human duodenal contents catalyzed the formation of ceramide from sphingomyelin most efficiently at slightly alkaline pH in the presence of conjugated bile salts. Under optimal conditions 1 ml duodenal contents hydrolyzed up to 300 nmole sphingomyelin per h. The hydrolysis of ceramide to sphingosine and free fatty acids and the reverse reaction were also catalyzed. The pH optimum of this reaction was about 7.6. That the enzymes are of intestinal origin was indicated by the data obtained when sphingomyelin and ceramide were incubated with rat pancreatic juice, pancreas homogenate and homogenate of pig intestinal mucosa. The preparations all had low sphingomyelinase activity at a pH of about 5, but only the pig mucosa homogenate had significant hydrolytic activity against sphingomyelin at alkaline pH and against oleoyl-sphingosine at pH 7.6. The latter enzymic activities were enriched in a brush-border preparation of rat intestinal mucosa to the same extent as the alkaline phosphatase activity. The brush border may therefore be the main site of hydrolysis of dietary sphingomyelin.

Effect of hog vitamin B12 binders on the uptake of vitamin B12 y tissue homogenates.

Two vitamin B12 binding components were separated from the hog intrinsic concentrate WES 942 by recycling gel filtration and examined for enhancing effect on the uptake of B12 by tissue homogenates. Only the minor component, B, which according to an earlier study possessed intrinsic factor identity, enhanced the uptake of B12 by guinea pig intestinal mucosa homogenate. Only the major B12 binding component, A, which had earlier been found to lack intrinsic factor identity, enhanced the uptake of B12 by rat-liver homogenate.

Effect of poly-L-arginine on rate of bovine IgG transport by newborn pig intestine.

MOVEMENT of protein molecules within and across the plasma membranes of mammalian cells probably occurs more widely than is generally realized. Examples of protein transport that are not normally considered in the same light include the supposed movement of “carrier” proteins within the plasma membrane1, the penetration of albumin into isolated tumour cells2 and the endocytosis of proteins by the epithelium of the small intestine3. Yet the protein–membrane interactions which probably accompany the movement of protein molecules in each case are likely to have certain features in common. For example, transported protein must show some temporary affinity for constituents of the membrane, and the actual movement of protein across the membrane will cause some distortion of otherwise stable structures. The experiments described here were designed to test whether the transport of protein by the pig intestinal mucosa was in any way similar to the transport of protein into tumour cells, a system which has been used extensively to study some of the macromolecular interactions associated with protein transport2.

Electrophoresis of acidic mucopolysaccharides in agarose gel.

A method of gel electrophoresis which separates polydisperse acidic mucopolysaccharides (AMPS) solely on the basis of differences in charge density is described. The standard procedure is to use a 0.6% agarose gel at pH 3.0 in the E-C 470 apparatus. The method shows that AMPS with the same basic polysaccharide chain structure, extracted from different tissues, may differ in electrophoretic mobility. It resolves heparin from pig intestinal mucosa into two major components.

Biosynthesis of Δ7-cholesten-3Β-ol, Δ5,7-cholestadien-3Β-ol, and Δ5-cholesten-3Β-ol by guinea pig intestinal mucosa in vitro

Methods were developed for the separation and determination of the various 27-carbon sterols of intestinal mucosa by means of thin-layer chromatography. Scrapings of the mucosa of the small intestine of guinea pig and rat were shown to incorporate isotope from (14)C-labeled acetate and mevalonate into sterols in vitro. For each substrate this activity was lowest in mucosa from the proximal third of the small intestine and greatest in mucosa from the more distal regions of the small intestine. The total 27-carbon sterol content of guinea pig mucosa varied only slightly along the length of the small intestine, but the concentration of cholesterol was highest distally. More than 95% of the radioactivity incorporated from acetate-2-(14)C into 27-carbon sterols by guinea pig mucosa in 4 hr was recovered as lathosterol and 7-dehydrocholesterol; less than 5% was in cholesterol. The specific activities of the 27-carbon sterols were consistent with the concept that synthesis proceeds from lathosterol to 7-dehydrocholesterol to cholesterol.

Intestinal dipeptidases. 3. Characterization and determination of dipeptidase activity in adult rat intestinal mucosa.

AbstractFive dipeptidase activities in the mucosa of the rat small intestine have been determined and characterized by the aid of a new spectrophotometric assay method.The distribution of the dipeptidase activities along the gastrointestinal tract has been investigated. The activities were low in the stomach, the cecum and the large intestine, while high activities were found along the whole length of the small intestine with the maximum localized to its middle part. The relation of our present findings to previous observations about the rat intestinal dipeptidases has been discussed. Some differences with respect to both their properties and their distribution were observed in com‐ parison with the dipeptidases of the pig intestinal mucosa.

EFFECT OF PEPTIDES ON THE UPTAKE OF CO57B12 BY THE GUINEA PIG INTESTINAL MUCOSA HOMOGENATES.

Over 30 di-, tri-, tetra-, and polypeptides derived from 14 aminoacids were assayed for their enhancing activity upon the uptake of radioactive B12 in the guinea pig intestinal homogenate system. No unequivocal activity of these materials could be demonstrated. The negative results obtained (including those with the use of glutamic acid) do not lend support to the concept that simple low-molecular-weight peptides or amino-acid units play a role in the intestinal absorption of Vitamin B12.

Intestinal dipeptidases I. Spectrophotometric determination and characterization of dipeptidase activity in pig intestinal mucosa

A new simple and rapid spectrophotometric assay for dipeptidase (dipeptide hydrolase, EC class 3.4.3) activity is described. The method is based upon the decrease of absorption at 220 μm as peptide bonds are hydrolyzed. By using ethanol as a precipitating reagent, the method is suitable for the study of peptidase activity in crude tissue extracts or partially purified preparations. The accuracy is about ± 1%, and the sensitivity is greatly increased as compared with procedures commonly used hitherto. The method has been employed for the determination and characterization of L-alanyl-L-glutamic acid, glycylglycine, glycyl-L-leucine and glycyl-L-valine hydrolysing activities of pig intestinal mucosa. The results suggest that the four different activities are related to four separate enzymes. The relation of our findings to previous knowledge of the properties of the dipeptidases has been discussed.

Comparative study of intrinsic factor activity of gastric materials by urinary excretion test in vivo and on guinea pig intestinal mucosa homogenates in vitro

Measurement of 57Co B 12 uptake by dialyzed and lyophilized guinea pig intestinal mucosa homogenates (GPIMH) is a sensitive in vitro test for the assay of human intrinsic factor activity. Whenever any of the juices or fractions processed from human gastric juice or mucosa was active on GPIMH, it was also invariably active on urinary excretion test in vivo. Conversely, materials known to be devoid of intrinsic factor activity, such as human serum, acidic serum glycoproteins, saliva and gastric juices from proven pernicious anemia patients, were inactive on GPIMH. Four anacid gastric juices from patients, after subtotal gastrectomy for gastric cancer, were also inactive on GPIMH. There was no straight line correlation between the IF activity of gastric juices in vivo on urinary excretion test, and in vitro on GPIMH. This was perhaps due to differences in the storage time of the acid gastric juices at the time of their in vivo and in vitro assays. Storage for several weeks in a frozen state caused IF activity loss in acid gastric juices, but it did not affect the anacid ones. The ratio of IF to the dose of 57Co B 12 in the system is also of significance in affecting the result of the GPIMH test. A discrepancy was noted in the activity of some of the hog mucosa IF preparations on GPIMH and urinary excretion test in vivo.

Assay of Intrinsic Factor activity on guinea pig intestinal mucosa homogenate.

Summary and conclusionsThe results of the assay of IF activity on fresh or dialyzed and lyophilized guinea pig intestinal mucosa homogenates (GPIMH) with the use of Co57B12 are reported. This method has been found suitable for assay of IF activity of native and lyophilized human gastric juices, extracts from human gastric mucosa, rat whole stomach and rat gastric mucosa. The prerequisite for significant enhancement of B12 uptake by the IF-containing materials is an optimal ratio of concentrations of IF to B12 and use of labeled vitamin of high specific activity. Heating of IF sources in boiling water, as well as alkalinization to the pH of 12 and above, reduces B12 uptake to levels obtainable with saline. While native human gastric juices from normal controls or duodenal ulcer patients showed a marked enhancement of B12 uptake by GPIMH, lyophilized gastric juices of 2 pernicious anemia patients showed very low IF activity. A gross correlation was found between results of the GPIMH assays and IF activity o...

On the conversion of fructose to glucose by guinea pig intestine

Inverted intestinal sacs of the guinea pig, but not of the rat, convert fructose to glucose during its passage through the intestinal wall. An exchange of 14C-activity between C-1 and C-6 of the glucose formed from [I- 14C]fructose under these conditions has been observed. Fructokinase and glucose-6-phosphatase have been found to be present in the intestinal mucosa of the guinea pig and it has been confirmed that the latter enzyme is absent in the same tissue of the rat. These observations lead to the conclusion that the conversion of fructose to glucose by the guinea pig intestinal mucosa occurs by the same mechanism as that operating in the liver, involving fructose-I-phosphate and triose phosphates as intermediates. It is suggested that the inability of rat intestine to convert fructose to glucose is due to the absence of glucose-6-phosphatase.